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The assay of vitamin D that began in the 1970s with the quantification of one or two metabolites, 25‐OH‐D or 1,25‐(OH)2D, continues to evolve with the emergence of liquid chromatography tandem mass spectrometry (LC‐MS/MS) as the technique of choice. This highly accurate, specific, and sensitive technique has been adopted by many fields of endocrinology for the measurement of multiple other components of the metabolome, and its advantage is that it not only makes it feasible to assay 25‐OH‐D or 1,25‐(OH)2D but also other circulating vitamin D metabolites in the vitamin D metabolome. In the process, this broadens the spectrum of vitamin D metabolites, which the clinician can use to evaluate the many complex genetic and acquired diseases of calcium and phosphate homeostasis involving vitamin D. Several examples are provided in this review that additional metabolites (eg, 24,25‐(OH)2D3, 25‐OH‐D3‐26,23‐lactone, and 1,24,25‐(OH)3D3) or their ratios with the main forms offer valuable additional diagnostic information. This approach illustrates that biomarkers of disease can also include metabolites devoid of biological activity. Herein, a case is presented that the decision to switch to a LC‐MS/MS technology permits the measurement of a larger number of vitamin D metabolites simultaneously and does not need to lead to a dramatic increase in cost or complexity because the technique uses a highly versatile tandem mass spectrometer with plenty of reserve analytical capacity. Physicians are encouraged to consider adding this rapidly evolving technique aimed at evaluating the wider vitamin D metabolome toward streamlining their approach to calcium‐ and phosphate‐related disease states. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Vitamin D receptor (VDR) null fetuses have normal serum minerals, parathyroid hormone (PTH), skeletal morphology, and mineralization but increased serum calcitriol, placental calcium transport, and placental expression of Pthrp, Trpv6, and (as reported in this study) Pdia3. We examined Cyp27b1 null fetal mice, which do not make calcitriol, to determine if loss of calcitriol has the same consequences as loss of VDR. Cyp27b1 null and wild-type (WT) females were mated to Cyp27b1+/- males, which generated Cyp27b1 null and Cyp27b1+/- fetuses from Cyp27b1 null mothers, and Cyp27b1+/- and WT fetuses from WT mothers. Cyp27b1 null fetuses had undetectable calcitriol but normal serum calcium and phosphorus, PTH, fibroblast growth factor 23 (FGF23), skeletal mineral content, tibial lengths and morphology, placental calcium transport, and expression of Trpv6 and Pthrp; conversely, placental Pdia3 was downregulated. However, although Cyp27b1+/- and null fetuses of Cyp27b1 null mothers were indistinguishable, they had higher serum and amniotic fluid calcium, lower amniotic fluid phosphorus, lower FGF23, and higher 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D than in WT and Cyp27b1+/- fetuses of WT mothers. In summary, loss of fetal calcitriol did not alter mineral or bone homeostasis, but Cyp27b1 null mothers altered mineral homeostasis in their fetuses independent of fetal genotype. Cyp27b1 null fetuses differ from Vdr null fetuses, possibly through high levels of calcitriol acting on Pdia3 in Vdr nulls to upregulate placental calcium transport and expression of Trpv6 and Pthrp. In conclusion, maternal calcitriol influences fetal mineral metabolism, whereas loss of fetal calcitriol does not. © 2018 American Society for Bone and Mineral Research.

BACKGROUND There are few controlled studies of the effect of different doses of vitamin D3 on bone mineral density (BMD). OBJECTIVES We conducted a randomized placebo-controlled trial of increasing doses of vitamin D3 in 163 Caucasian and 31 African American women with serum 25-hydroxyvitamin D (25OHD) ≤50 nmol/L. This is an analysis of secondary outcome BMD to see if there is an association between percent change in BMD and dose of vitamin D3. METHODS Participants were randomly assigned to placebo, vitamin D3 400, 800, 1600, 2400, 3200, 4000, or 4800 IU/day; calcium supplements, average 600mg, were given to provide a total calcium intake of 1200 mg/d. The primary outcome was 12-month serum 25OHD level. Analysis methods include ANOVA and Pearson correlations. RESULTS The mean percent increase (±SD) in BMD at 12 months for all women was small; total body, 0.62% (± 2.72), femoral neck 0.59% (±3.58) and spine 0.43% (±2.80). There was no difference in BMD or serum N-telopeptide in response to vitamin D by dose or race. The increase in total body, spine and hip BMD in elderly women given vitamin D doses between 400 and 4800 IU daily and calcium supplementation is small, unrelated to dose or 12-month serum 25OHD, free 25OHD or 1,25(OH)2D. There was no evidence of a threshold change in BMD with increasing serum 25OHD or free 25OHD in this population. CONCLUSIONS We found no significant effect of daily vitamin D dose ranging from 400 to 4800 IU/day on BMD or serum N-terminal telopeptides in elderly women with initially low serum 25OHD.

Vitamin D requires a two-step activation by hydroxylation: The first step is catalyzed by hepatic 25-hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α-hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25-(OH)2 D. Mutations of CYP2R1 have been associated with vitamin D-dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss-of-function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss-of-function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25-OH-D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25-(OH)2 D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa-hydroxyvitamin D3 or alfacalcidol (1α-OH-D3 ) treatment, and we observed a dramatic increase in the 1,25-(OH)2 D serum concentration, which indicated the role of accessory 25-hydroxylase enzymes. Lastly, in patients who received calcifediol (25-OH-D3 ), we documented normal 24-hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25-OH-D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research.

CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3 :24,25-(OH)2 D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2 D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2 D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2 D3 as a contaminant of the 24,25-(OH)2 D3 peak. In normals, the concentration of 24,25-(OH)2 D3 greatly exceeds 25,26-(OH)2 D3 ; however, 25,26-(OH)2 D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2 D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2 D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2 D3 from 25,26-(OH)2 D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2 D3 in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.

The roles of vitamin D, mediated through its conversion to 1,25-dihydroxyvitamin D(3) (calcitriol), have been expanded recently through new knowledge about the range of tissues capable of activating it and the breadth of the genes under its regulatory control. This basic science together with the fact that numerous studies across North America are revealing that vitamin D insufficiency/deficiency (as defined by 25-OH-D levels 80%) in dialysis patients are indicative that these patients have two vitamin D-related problems that require different treatment regimens. Combinations of vitamin D(3) (cholecalciferol) or vitamin D(2) (ergocalciferol) and an active calcitriol analog should be used to treat their vitamin D deficiency and their calcitriol hormone insufficiency, respectively. This mini-review provides the case for combination therapy.

We determined the relationship between bone mass and age, anthropometric variables, creatinine clearance (Ccr), and serum and urine biochemical variables in 77 normal white women (aged 41-86, mean = 67) living in their own homes. A total of 74 women were postmenopausal. Skeletal status was assessed in all subjects by x-rays of the hand with measurement of the mean combined cortical thickness (CCT) of the second metacarpal bones. In 53 women, bone mineral content of the radial shaft (RMBC) was also measured by single-photon absorptiometry (SPA) and lumbar bone mineral density (LBMD) was measured by dual-photon absorptiometry (DPA). Serum and urine biochemical variables were measured under standardized conditions on the sixth and seventh days of a controlled diet. There was a strong positive correlation between Ccr and bone mass. Although our subjects showed the expected linear decline in Ccr with age, we found that the relationship between Ccr and bone mass in the radius and lumbar spine was independent of age. On the other hand, the relationship between Ccr and CCT was not independent of age. We concluded that the relationship between Ccr and lumbar and radial bone mass is probably indicative of a relationship between glomerular filtration rate and bone mass, although this requires validation with a noncreatinine method for measurement of glomerular filtration rate. Age per se does not appear to be a cause of declining lumbar bone mass after the menopause.

The metabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10(-11) M) and pharmacologic (3.5 x 10(-7) M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10(-7) M 1,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spectrometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from 1,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-1,25-dihydroxyvitamin D3; 24-oxo-1,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. Evidence is presented that (1) 1,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11(-11) M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10(-7) M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.

Summary Objective To compare biochemical variables, renal function and calcium and vitamin D intakes in euparathyroid and hyperparathyroid patients with primary osteoporosis and osteopenia and describe the measures necessary to normalize serum PTH in the patients with secondary hyperparathyroidism. Design and patients We reviewed the charts of normocalcemic patients with primary osteoporosis and osteopenia first seen during the years 1991–2003 and identified 75 with elevated serum PTH levels at baseline. These patients were compared to all the 143 euparathyroid patients first seen in 1998 and 1999. Patients were restudied after 1 year and we attempted to follow patients with secondary hyperparathyroidism until PTH levels became normal. Measurements At baseline serum PTH, ionized calcium, inorganic phosphate, alkaline phosphatase, creatinine, a complete blood count and serum 25 hydroxy vitamin D were measured in the early morning fasting state. These tests were repeated at follow up. Results In one-third of the hyperparathyroid patients, the standard baseline treatment failed to correct the secondary hyperparathyroidism necessitating extraordinary measures including unusually large doses of vitamin D (i.e. 50 000 IU vitamin D2 twice weekly) or the substitution of calcium citrate for calcium carbonate as a calcium supplement. Conclusion Large doses of vitamin D are frequently necessary to suppress secondary hyperparathyroidism in patients with primary osteoporosis and osteopenia. This suggests that vitamin D metabolism may be altered in some of these patients.

Plasma 25(OH)D has emerged as a valuable biomarker for the many varied health-related effects of vitamin D in the clinic mainly because of the recognition of the importance of the enzyme, CYP27B1, or the 25(OH)D-alpha-hydroxylase in the extrarenal, target cell production of calcitriol. This review briefly assesses current methodology for plasma 25(OH)D assay focusing mainly on currrent controversies surrounding the definition of the normal range and performance characteristics of the assay, separate measurement of both 25(OH)D(2) and 25(OH)D(3), and quality assurance tesing of laboratories offering the test. Clinicians have two main types of 25(OH)D assay based on either high-performance liquid chromatography with UV or mass detection or higher throughput kits based on protein (competitive protein binding assay or radioimmunoassay) binding. Based on 30 yr of experience with measuring 25(OH)D levels, it is concluded that, in the hands of appropriately trained experts, both types of assay provide reliable and accurate results, but all laboratories providing 25(OH)D data need frequent external quality assurance service to ensure that this performance is maintained.

Low 25-hydroxyvitamin D3 (25-OH-D3) concentration in pregnancy increases the risk of preeclampsia (PE) based on studies from countries with vitamin D fortification policy. The objective of this stu...

OBJECTIVE Vitamin D 3 (D 3 ) is not active but must be hydroxylated at C-25 in liver before acquiring its hormonal potential in the kidney. The sterol-27 hydroxylase (gene symbol: CYP27A) catalyses the oxidation of sterol side chain in bile acid synthesis but the enzyme is also known as a D 3 -25 hydroxylase. DESIGN The study examined the expression of the gene encoding CYP27A in adult and fetal human livers and kidneys. SUBJECTS Thirty-nine adults (18 men and 21 women; mean age 58 years in men and 57 years in women) and three normal fetuses gestational age 17-19 weeks were studied. MEASUREMENTS Tissue CYP27A mRNA and serum 250HD concentrations were measured. RESULTS Normal specimens: CYP27A transcript was found to be higher In adult than in fetal livers but its expression was similar in adult and fetal kidneys. In fetuses, no difference was observed between CYP27A levels in livers and kidneys. In adult livers CYP27A levels were higher in women than in men. Hepatic CYP27A mRNA and serum 250HD concentrations were both found to be higher in summer than in winter. Multiple linear regression analyses indicate that the season of the year and the serum 250HD concentrations (but not 1,25(OH) 2 D concentrations) are the best predictors of CYP27A mRNA abundance in normal adult livers. In situ hybridization illustrates a clear label in hepatocytes which increases in intensity in the perivenous region of the hepatic acinus. Pathological specimens: In one man with an hepatic carcinoma there was a very large increase in CYP27A (> 1000 fold) compared to the level found in the normal liver. In that patient, serum 250HD concentrations were found to be high considering the level of CYP27A mRNA in the normal hepatic area suggesting that the neoplastic tissue contributed to the C-25 hydroxylation of vitamin D. Specimens obtained from two patients suffering from focal hepatic hyperplasia indicate that in one case the level of CYP27A mRNA was twice as high in the pathological than in the normal area while in the other its levels were similar in both areas. No difference in the CYP27A transcript was observed between specimens obtained from normal areas and those obtained form either an hepatic adenoma or from two intrahepatic colonic metastases. CONCLUSIONS CYP27A is present not only in the human adult liver but also in the adult kidney, and in the fetal liver and kidney. Our findings illustrate that CYP27A can be significantly upregulated in certain pathological situations such as in hepatic carcinoma and that the neoplastic tissue could contribute to the circulating concentration of 250HD.

Previous studies have suggested that both plasma 24,25-dihydroxyvitamin D [24,25-(OH)2D] concentrations and renal 25-hydroxyvitamin D-24-hydroxylase activity are increased in mice with X-linked hypophosphatemia (Hypmice). However, because the plasma levels of 24,25-(OH)2D seemed surprisingly high, we repeated these assays using two different techniques. Mass fragmentographic and radioreceptor assays were employed to compare the plasma concentrations of 25-hydroxyvitamin D (25-OHD) and 24,25-(OH)2D in normal mice with those inHypmice. These assays yielded 24,25-(OH)2D concentrations much lower than previously reported in mice (both normal andHyp). The concentrations of 25-OHD3, and 24,25-(OH)2D3, determined by mass fragmentography, were lower inHypmice than in controls [25-OHD3, 9.7 ± 0.4 versus 14.6 ± 0.6 ng/ml, p < 0.01; 24,25-(OH)2D3, 7.1 ± 0.3 versus 10.4 ± 0.4 ng/ml, p < 0.01]. Plasma 25-OHD concentration was the main determinant of plasma 24,25-(OH)2D, and the ratio of 25-OHD3 to 24,25-(OH)2D3 obtained from mass fragmentographic measurements did not differ between the two groups (1.40 ± 0.05 versus 1.36 ± 0.03 ng/ml, NS in normal andHypgroups, respectively). Separate measurement of plasma 25-OHD, 24,25-(OH)2D, and 25-OHD3-26,23-lactone by radioreceptor assay showed no difference between either plasma 24,25-(OH)2D, or the ratio of 25-OHD concentration to 24,25-(OH)2D concentration amongHypand control animals. In neither study was plasma phosphate concentration related to the 25-OHD3: 24,25-(OH)2D3 ratio. We conclude that previous estimations of plasma 24,25-(OH)2D3 concentrations in mice were erroneously high and, further, that in fact this metabolite is not present in increased concentration inHyp mice